Endocrine disrupting chemical (EDC)-induced transgenerational alterations in DNA methylation, also called epimutations, have been demonstrated in mammals and fish. It is not yet clearly understood if somatic cells can inherit such transgenerational epimutations from germline and whether the epimutations can be corrected by CRISPR-dCas9 epigenome editing tools in vivo. Fish are excellent models to develop such tools for two reasons- a) EDC-induced phenotypes and epimutations have been demonstrated in fish models, and 2) CRISPR-Cas9 genome editing tools can be used more efficiently because of their external embryonic development and transparency. In two independent studies, we have found transgenerational phenotypes with 20-30% reduced fertility in the males at the third generation (F2) after exposure of embryos during the first generation (F0) to the pharmaceutical estrogen, 17a-ethinylestradiol (EE2, a model EDC). These observations suggest that embryonic EE2 exposure alters programming of developing germ cells leading to transgenerational male subfertility phenotype. The male germ cells from EE2-exposed fish maintained global hypomethylation including DNA methyltransferase 1 (Dnmt1) expression at a suppressed state at both F0 and F2 generation. We, therefore, hypothesized that EE2 induces hypomethylation in germ cells' genome at F0 generation which is inherited by F2 generation germ cells and soma resulting in alterations of transcriptional networks and gene expression leading to reproductive impairment in male gonads. Since the effects were mediated by male germ line, we propose to identify EE2-induced transgenerational epimutations in males by whole genome bisulfite sequencing (WGBS) and to correct phenotype-specific epimutations in vivo by CRISPR-dCas9 genome editing method. The proposed study has two specific aims. Aim 1 will identify EE2- induced genome-wide epimutations in F0 and F2 generations. EE2-induced epimutations will be analyzed in F0 primordial germ cells and sperm and in F2 sperm and testicular somatic cells by WGBS. Unique epimutations that were present in F0 generation and that are associated with F2 male reproductive impairment will be selected for genome editing. Aim 2 will remove epimutations (DNA methylation or demethylation marks) by CRISPR-dCas9 tools to recover a reproductively healthy phenotype at F2 and F3 generations using embryos from the F1 and F2 parental lineages. We will microinject programmable CRISPR-Tet1-dCas9 or CRISPR-Dnmt3a-dCas9 or CRISPR-Dnmt1-dCas9 into F2 and F3 zygotes at the 1-cell stage. Resulting adult males will be tested for recovery of reproductive function. Results from this proposed R21 study will be used to develop a R01 project directed toward development of epigenome editing tools to correct transgenerational phenotypes in vivo in other model organisms. Results from this study provide bring new insights into epigenetic mechanisms underlying transgenerational diseases in humans.